The present invention is directed to modified hemoglobin-like compounds, and more particularly to modified hemoglobin-like polypeptides and proteins. The present invention is directed also to methods of purifying such modified hemoglobin-like compounds.
Hemoglobin (referred to herein as "Hb") is the oxygen-carrying component of blood. Hemoglobin circulates through the bloodstream inside small enucleate cells called erythrocytes (red blood cells). Hemoglobin is a protein constructed from four associated polypeptide chains, and bearing prosthetic groups known as hemes. The erythrocyte helps maintain hemoglobin in its reduced, functional form. The heme iron atom is susceptible to oxidation, but may be reduced again by one of two enzyme systems within the erythrocyte, the cytochrome b.sub.5 and glutathione reduction systems.
Hemoglobin binds oxygen at a respiratory surface (skin, gills, trachea, lung, etc.) and transports the oxygen to inner tissues, where it is released and used for metabolism. In nature, low molecular weight hemoglobins (16-120 kilodaltons) tend to be enclosed in circulating red blood cells, while the larger polymeric hemoglobins circulate freely in the blood or hemolymph.
The structure of hemoglobin is well known as described in Bunn & Forget, eds., Hemoglobin: Molecular, Genetic and Clinical Aspects (W. B. Saunders Co., Philadelphia, Pa.: 1986) and Fermi & Perutz "Hemoglobin and Myoglobin," in Phillips and Richards, Atlas of Molecular Structures in Biology (Clarendon Press: 1981).
About 92% of normal adult human hemolysate is Hb A.sub.o (designated alpha.sub.2 beta.sub.2 because it comprises two alpha and two beta chains). In a hemoglobin tetramer, each alpha subunit is associated with a beta subunit to form a stable alpha/beta dimer, two of which in turn associate to form the tetramer. The subunits are noncovalently associated through Van der Waals forces, hydrogen bonds and salt bridges. The amino add sequences of the alpha and beta globin polypeptide chains of Hb A.sub.o are given in Table 1 of PCT Publication No. WO 93/09143. The wild-type alpha chain consists of 141 amino acids. The iron atom of the heme (ferroprotoporphyrin IX) group is bound covalently to the imidazole of His 87 (the "proximal histidine"). The wild-type beta chain is 146 residues long and heme is bound to it at His 92.
The human alpha and beta globin genes reside on chromosomes 16 and 11, respectively. Bunn and Forget, infra at 172. Both genes have been cloned and sequenced, Liebhaber, et al., PNAS 77: 7054-58 (1980) (alpha-globin genomic DNA); Marotta, et al., J. Biol. Chem., 252:5040-53 (1977) (beta globin cDNA); Lawn, et al., Cell, 21:647 (1980) (beta globin genomic DNA).
Hemoglobin exhibits cooperative binding of oxygen by the four subunits of the hemoglobin molecule (the two alpha globins and two beta globins in the case of Hb A.sub.o), and this cooperativity greatly facilitates efficient oxygen transport. Cooperativity, achieved by the so-called heme-heme interaction, allows hemoglobin to vary its affinity for oxygen. Cooperativity can also be determined using the oxygen dissociation curve (described below) and is generally reported as the Hill coefficient, "n" or "n.sub.max." Hemoglobin reversibly binds up to four moles of oxygen per mole of hemoglobin.
Oxygen-carrying compounds are frequently compared by means of a device known as an oxygen dissociation curve. This curve is obtained when, for a given oxygen carrier, oxygen saturation or content is graphed against the partial pressure of oxygen. For Hb, the percentage of saturation increases with partial pressure according to a sigmoidal relationship. The P.sub.50 is the partial pressure at which the oxygen-carrying species is half saturated with oxygen. It is thus a measure of oxygen-binding affinity; the higher the P.sub.50, the more readily oxygen is released.
The ability of hemoglobin to alter its oxygen affinity under physiological conditions, increasing the efficiency of oxygen transport around the body, is largely dependent on the presence of the metabolite 2,3-diphosphoglycerate (2,3-DPG). The oxygen affinity of hemoglobin is lowered by the presence of 2,3-DPG. Inside the erythrocyte 2,3-DPG is present at a concentration nearly as great as that of hemoglobin itself. In the absence of 2,3-DPG "conventional" hemoglobin (hemoglobin A.sub.o) binds oxygen very strongly at physiological oxygen partial pressures and would release little oxygen to respiring tissue. Accordingly, any substitute for hemoglobin must somehow correct the oxygen affinity and/or the Hill coefficient to physiologically meaningful levels (see e.g., Rausch, C. and Feola, M., U.S. Pat. Nos. 5,084,558 and 5,296,465; Sehgal, L. R., U.S. Pat. Nos. 4,826,811 and 5,194,590; Hoffman et al., WO 90/13645; Hoffman and Nagai, U.S. Pat. No. 5,028,588; Anderson et al., WO 93/09143; Fronticelli, C. et al., U.S. Pat. No. 5,239,061; and De Angelo et al., WO 93/08831 and WO 91/16349).
It is not always practical or safe to transfuse a patient with donated blood. In these situations, use of a red blood cell ("RBC") substitute is desirable. When human blood is not available or the risk of transfusion is too great, plasma expanders can be administered. However, plasma expanders, such as colloid and crystalloid solutions, replace only blood volume, and not oxygen carrying capacity. In situations where blood is not available for transfusion, a red blood cell substitute that can transport oxygen in addition to providing volume replacement is desirable.
To address this need, a number of red blood cell substitutes have been developed (Winslow, R. M.(1992) Hemoglobin-based Red Cell Substitutes, The Johns Hopkins University Press, Baltimore 242 pp). These substitutes include synthetic perfluorocarbon solutions, (Long, D. M. European Patent 0307087), stroma-free hemoglobin solutions, both chemically crosslinked and uncrosslinked, derived from a variety of mammalian red blood cells (Rausch, C. and Feola, M., U.S. Pat. Nos. 5,084,558 and 5,296,465; Sehgal, L. R., U.S. Pat. Nos. 4,826,811 and 5,194,590; Vlahakes, G. J. et al., (1990) J. Thorac. Cardiovas. Surg. 100: 379-388) and hemoglobins expressed in and purified from genetically engineered organisms (for example, non-erythrocyte cells such as bacteria and yeast, Hoffman et al., WO 90/13645; bacteria, Anderson et al., WO 93/09143, bacteria and yeast Fronticelli, C. et al., U.S. Pat. No. 5,239,061; yeast, De Angelo et al., WO 93/08831 and WO 91/16349; and transgenic mammals, Logan et al., WO 92/22646; Townes, T. M and McCune, S. L., WO 92/11283). These red blood cell substitutes have been designed to replace or augment the volume and the oxygen carrying capability of red blood cells.
However, red blood cell replacement solutions that have been administered to animals and humans have exhibited certain adverse events upon administration. These adverse reactions have included hypertension, renal failure, neurotoxicity, and liver toxicity (Winslow, R. M., (1992) Hemoglobin-based Red Cell Substitutes, The Johns Hopkins University Press, Baltimore 242 pp.; Biro, G. P. et al., (1992) Biomat., Art. Cells & Immob. Biotech. 20: 1013-1020). In the case of perfluorocarbons, hypertension, activation of the reticulo-endothelial system, and complement activation have been observed (Reichelt, H. et al., (1992) in Blood Substitutes and Oxygen Carriers, T. M. Chang (ed.), pg. 769-772; Bentley, P. K. supra, pp. 778-781). For hemoglobin-based oxygen carriers, renal failure and renal toxicity are the result of the formation of hemoglobin .alpha./.beta. dimers. The formation of dimers can be prevented by chemically crosslinking (Sehgal, et al., U.S. Pat. Nos. 4,826,811 and 5,194,590; Walder, J. A. U.S. Reissue Pat. RE34271) or genetically linking (Hoffman, et al, WO 90/13645) the hemoglobin dimers so that the tetramer is prevented from dissociating.
Prevention of dimer formation has not alleviated all of the adverse events associated with hemoglobin administration. Blood pressure changes and gastrointestinal effects upon administration of hemoglobin solutions have been attributed to vasoconstriction resulting from the binding of endothelium derived relaxing factor (EDRF) by hemoglobin (Spahn, D. R. et al., (1994) Anesth. Analg. 78: 1000-1021; Biro, G. P., (1992) Biomat., Art. Cells & Immob. Biotech., 20: 1013-1020; Vandegriff, K. D. (1992) Biotechnology and Genetic Engineering Reviews, Volume 10: 404-453 M. P. Tombs, Editor, Intercept Ltd., Andover, England). Endothelium derived relaxing factor has been identified as nitric oxide (NO) (Moncada, S. et al., (1991) Pharmacol. Rev. 43: 109-142 for review); both inducible and constitutive NO are primarily produced in the endothelium of the vasculature and act as local modulators of vascular tone.
When hemoglobin is contained in red blood cells, it cannot move beyond the boundaries of blood vessels. Therefore, nitric oxide must diffuse to the hemoglobin in an RBC before it is bound. When hemoglobin is not contained within an RBC, such as is the case with hemoglobin-based blood substitutes, it may pass beyond the endothelium lining the blood vessels and penetrate to the extravascular space (extravasation). Thus, a possible mechanism causing adverse events associated with administration of extracellular hemoglobin may be excessive inactivation of nitric oxide due to hemoglobin extravasation. Furthermore, NO is constitutively synthesized by the vascular endothelium. Inactivation of NO in the endothelium and extravascular space may lead to vasoconstriction and the pressor response observed after infusions of cell-free hemoglobin. Larger hemoglobins may serve to reduce hypertension associated with the use of some extracellular hemoglobin solutions.
In addition to the effects noted above, the dosage of non-polymeric extracellular hemoglobin that can be administered may be limited by the colloidal osmotic pressure (COP) of the solution. Administration of an extracellular hemoglobin composed of hemoglobin tetramers that would have the same grams of hemoglobin as a unit of packed red blood cells might result in a significant influx of water from the cells into the blood stream due to the high colloid osmotic pressure of the hemoglobin solution. Polymeric hemoglobin solutions can be administered at higher effective hemoglobin dosages, because as the molecular weight increases, the number of the individual molecules is decreased, resulting in reduced COP (Winslow, R. M., (1992) Hemoglobin-based Red Cell Substitutes, The Johns Hopkins University Press, Baltimore, pp 34-35).
Some higher molecular weight hemoglobins occur in nature. For example, there are three mutants of human hemoglobin that are known to polymerize as a result of formation of intermolecular (first tetramer to second tetramer) disulfide bridges. Tondo, Biochem. Biophys. Acta, 342:15-20 (1974) and Tondo, An. Acad. Bras. Cr., 59:243-251 (1987) describe one such mutant known as Hb Porto Alegre. Hb Mississippi is characterized by a cysteine substitution in place of Ser CD3(44).beta. and is believed to be composed of ten or more hemoglobin tetramers according to Adams et al., Hemoglobin, 11(5):435-542 (1987). Hemoglobin Ta Li is characterized by a .beta.83(EF7)Gly.fwdarw.Cys mutation, which showed slow mobility in starch gel electrophoresis, indicating that it too was a polymer.
There are a few known naturally occurring mutants of non-polymerizing human hemoglobins that have a cysteine mutation that do not polymerize (Harris et al., Blood, 55(1):131-137 (1980)(Hemoglobin Nigeria); Greer et al., Nature [New Biology], 230:261-264 (1971) (Hemoglobin Rainier). Hemoglobin Nunobiki (.alpha. 141 Arg.fwdarw.Cys) also features a non-polymerizing cysteine substitution. In both Hb Rainier and Hb Nunobiki, the mutant cysteine residues are surface cysteines.
Polymeric hemoglobins have also been reported in various vertebrates and invertebrates. Murine polymeric hemoglobins are described in Bonaventura & Riggs (Science, (1967)149:800-802) and Riggs (Science, (1965)147:621-623). A polymerizing hemoglobin variant in macaque monkeys is reported in Takenaka et al., Biochem Biophys. Acta, 492:433-444 (1977); Ishimoto et al., J. Anthrop. Soc. Nippon, 83(3):233-243 (1975). Both amphibians and reptiles also possess polymerizing hemoglobins (Tam et al., J. Biol. Chem., (1986) 261:8290-94).
Some invertebrate hemoglobins are also large multi-subunit proteins. The extracellular hemoglobin of the earthworm (Lumbricus terrestris) has twelve subunits, each of which is a dimer of structure (abcd).sub.2 where "a", "b", "c", and "d" denote the major heme containing chains. The "a", "b", and "c" chains form a disulfide-linked trimer. The whole molecule is composed of 192 heme-containing chains and 12 non-heme chains, and has a molecular weight of 3800 kDa. The brine shrimp Artemia produces three polymeric hemoglobins with nine genetically fused globin subunits (Manning, et al., Nature, (1990) 348:653). These are formed by variable association of two different subunit types, a and b. Of the eight intersubunit linkers, six are 12 residues long, one is 11 residues and one is 14 residues.
Non-polymerizing crosslinked hemoglobins have been artificially produced. For example, hemoglobin has been altered by chemically crosslinking the alpha chains between the Lys99 of alpha.sub.1 and the Lys99 of alpha.sub.2 (Walder, U.S. Pat. Nos. 4,600,531 and 4,598,064; Snyder, et al., PNAS (USA) (1987) 84: 7280-84; Chatterjee, et al., J. Biol. Chem., (1986) 261: 9927-37). The beta chains have also been chemically crosslinked (Kavanaugh, et al., Biochemistry, (1988) 27: 1804-8). U.S. Pat. No. 5,028,588 suggests that the T state of hemoglobin (corresponding to deoxygenated hemoglobin) may be stabilized by intersubunit (but intratetrameric) disulfide crosslinks resulting from substitution of cysteine residues for other residues.
Hemoglobin has also been artificially crosslinked to form polymers. For example, U.S. Pat. No. 4,001,401, U.S. Pat. No. 4,001,200, U.S. Pat. No. 4,777,244 and U.S. Pat. No. 4,053,590 all relate to polymerization of red blood cell-derived hemoglobin by chemical crosslinking. The crosslinking is achieved with the aid of bifunctional or polyfunctional crosslinking agents, especially those reactive with exposed amino groups of the globin chains. Aldehydes such as glutaraldehyde and glycolaldehyde have been used to crosslink hemoglobin both intramolecularly (within a tetramer) and intermolecularly (between tetramers). Intramolecular crosslinks serve to prevent dimerization into alpha/beta dimers and may also alter oxygen affinity, while intermolecular crosslinks create polymers of tetrameric hemoglobin. Polymeric hemoglobins may result in reduced extravasation because of their increased size. Reduced extravasation may, in turn, lead to reduced pressor effects resulting from infused hemoglobin solutions.
The result of these polymerization chemistries that have been used to crosslink hemoglobins is a polydisperse composition of covalently crosslinked aggregates. Bucci, U.S. Pat. No. 4,584,130, at col. 2, comments that "the polyhemoglobin reaction products are a heterogeneous mixture of various molecular species which differ in size and shape. The molecular weights of these polyhemoglobins range from 64,500 to 600,000 Daltons. The separation of individual molecular species from the heterogeneous mixture is virtually impossible. In addition, although longer retention times in vivo are obtained using polyhemoglobins, the oxygen affinity thereof is higher than that of stroma-free hemoglobin."
It is well recognized that random polymerization is difficult to control and that a number of different polymers can be obtained, commonly between two and ten tetramers per polymer. For example, according to Tye, U.S. Pat. No. 4,529,179, polymerized pyridoxylated hemoglobin has "a profound chemical heterogeneity making it difficult to study as a pharmaceutical agent."
Furthermore, once hemoglobin is polymerized, purification of specific molecular weight fractions can be accomplished using only molecular weight separation techniques. For example, tangential flow separation techniques can be used to separate certain size ranges of polymerized hemoglobins. However the membranes that are available for such separations are available only in a limited number of size ranges which allow the production of hemoglobins less than 100 kDa or greater than 300 kDa. In addition, such membranes are cumbersome, expensive, difficult to clean and the separation can be very slow.
Size exclusion chromatography (also known as, for example, gel filtration chromatography or gel permeation chromatography) has also been used in the past to separate hemoglobin molecular weight fractions. However, this technique is not suitable for large scale operation, and furthermore, does not provide good resolution for separation of molecular weight fractions (Simoni et al., (1993) Anal. Chim. Acta, 279: 73-88).
Simoni (1993, infra) also report the use of ion exchange chromatography to separate different molecular weight fractions of hemoglobin polymers. However, these workers noted that this kind of separation required differences in net charges. In addition, they used a salt gradient elution to separate the different molecular weight fractions, and they did not demonstrate any significant resolution of tetramer, octamer and decamer.
Correlations of molecular weight with serum half life for various proteins, such as IL-2, demonstrate that a significantly longer half life may be expected as the molecular weight of a protein increases, particularly above the renal filtration limit of 50-70 kDa. The use of crosslinkers that can inhibit the degradation of hemoglobin tetramers into dimers that are readily cleared can also lead to increased serum half life.
Accordingly, a need exists for additional hemoglobin-like compounds having these desired characteristics. In addition, a need exists for simple methods of creating specific molecular weight distributions in high molecular weight hemoglobin mixtures. The present invention satisfies these needs and provides related advantages.